The present invention relates to methods for extracting hemoglobin from erythrocytes and purifying hemoglobins. The methods of the present invention are particularly suitable for the extraction and purification of hemoglobin from erythrocytes on a commercial scale.
Advances have occurred in recent years in the development of hemoglobin-based blood substitutes. Such transfusional fluids serve as alternatives to whole blood or blood fractions for use as oxygen carriers and plasma expanders.
The use of whole blood and fractions thereof has grown increasingly disfavored because of the risk of immune or non-immune reactions and infections, such as acquired immunodeficiency syndrome. Even if it were possible to reduce these risks to an acceptable level, a need would still exist for non-native hemoglobin-based blood substitutes because of the chronic short supply of human-based products. To meet the demand for transfusional fluids, researchers have sought to develop a hemoglobin-based blood substitute free of the risks associated with whole blood and whole blood products.
Initial efforts were directed to developing hemoglobin solutions free of stromal components. Stromal components had been identified as a cause of coagulopathy and associated renal failure. Rabiner et al., J. Exp. Med., 126, 1127 (1967), used centrifugation and ultrafiltration procedures to prepare stroma-free hemoglobin solutions. Stroma-free hemoglobin solutions were prepared by re-crystallization by DeVenuto et al., J. Lab. Clin. Med., 89, 509 (1977).
The need to remove stromal phospholipids from hemoglobin-based blood substitutes is well-known. See, e.g., Bolin et al., "Advances In Blood Substitute Research," Prog. Clin. Biol. Res., 122, 117 (1983). In addition, hemoglobin-based blood substitutes must have a low oxygen affinity and a long transfusion half-life, and be substantially free of endotoxins, DNA and non-heme proteins and polypeptides.
Stromal contamination results from proteolysis during the separation of hemoglobin from erythrocytes. In PCT Patent Application No. WO 91US/09615, this problem was addressed by dialyzing the erythrocytes under slightly hypoosmotic conditions that rendered the cell membranes permeable to hemoglobin with a minimum of lysis. Nevertheless, unwieldy multiple chloroform extractions and centrifugations were still needed to reduce the stromal phospholipids to safe levels.
Endotoxins are found as a result of bacterial contamination. A synergistic toxicity between hemoglobin and endotoxin was recognized as early as 1963 by Litwin et al., Ann. Surg., 157(4), 485 (1963). White et al., J. Lab. Clin. Med., 108(2), 121 (1986) reported that stroma-free hemoglobin solutions purified to less than 0.12 EU/mL (120 picograms endotoxin per milliliter) produced cardiac rhythm disturbances and coagulation abnormalities. Heretofore, such endotoxin levels were ordinarily considered acceptable for pharmaceutical compositions for large volume parenteral administration.
U.S. Pat. No. 5,084,558 discloses the separation of phospholipids and endotoxins from hemoglobin solutions by High Performance Liquid Chromatography (HPLC) using a quaternary amine anion exchange medium on a silica support matrix. The three components are separated by an elution gradient, with release of the phospholipids taking place prior to the elution of the hemoglobin, and the endotoxins eluting after the hemoglobin. Simoni et al., in Anal. Chim. Acta., 249, 169 (1991) attributed the residual toxicity of endotoxin and phospholipid purified hemoglobin solutions to non-heme peptides and other proteins, which are not separated by any single anion-exchange liquid chromatography medium. A commercially impractical purification method using a combination of different anion-exchange liquid chromatography columns was also suggested.
The purification methods disclosed to date have also failed to provide for the large-scale commercial extraction and purification of hemoglobin. HPLC procedures are limited by the size of available columns and flow rates that are normally measured in milliliters rather than liters per minute. See, e.g., Hedlund et al., "Advances in Blood Substitute Research," Prog. Clin. Biol. Res., 122, 71 (1983). This is particularly true of affinity chromatography endotoxin separation techniques, which use chromatography columns containing 1 milliliter of separation media.
Thus, there remains a need for commercially feasible methods for the large-scale commercial extraction and purification of hemoglobin.